Directing sequence-specific proteolysis to new targets. The influence of loop size and target sequence on selective proteolysis by tissue-type plasminogen activator and urokinase-type plasminogen activator.
We have previously used substrate phage display to identify peptide sequences that are efficiently and selectively cleaved by tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). We demonstrate that this information can be used to direct selective proteolysis to new protein targets. Sequences that were labile to selective cleavage by t-PA or u-PA when in the context of a peptide were introduced into the 43-52 (or Omega) loop of staphylococcal nuclease. Both t-PA and u-PA hydrolyze the engineered proteins at the inserted target sequences, and Km values for protein cleavage were reduced up to 200-fold relative to values for cleavage of analogous sequences within 15 residue peptides. Variation of loop size surrounding a target sequence affects the efficiency of t-PA approximately 5-fold more strongly than that of trypsin, suggesting that cleavage by t-PA is more dependent on target site mobility. Cleavage of proteins by t-PA and u-PA is sequence selective. u-PA is 47-fold more active than t-PA for cleavage of a sequence known to be u-PA selective within small peptide substrates, whereas t-PA is 230-fold more active toward a t-PA-selective sequence.
Coombs
G S
GS
Department of Pharmacology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
Bergstrom
R C
RC
Madison
E L
EL
Corey
D R
DR
D002384:Catalysis; D006868:Hydrolysis; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000378:metabolism*
D008836:Micrococcal Nuclease; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator